Prap1在神经内分泌胃癌细胞对RAD001耐药过程中的作用及其Mad1相关调控机制研究

For many tumor types, neuroendocrine phenotype indicates higher malignancy and worse clinical prognosis. Although the mTOR inhibitors, represented by RAD001 (Everolimus), can significantly inhibit the proliferation of neuroendocrine tumor cells in vitro and prolong the progression free survival of the patients in clinical trails, the resistance of the tumor cells induced by the treatment impairs the anti-tumor effects in vivo. Up to now, the resistance mechanism of neuroendoctine tumor cells to mTOR inhibitors has not been fully illustrated. We have previously screened the resistance-related genes based on a new mouse model system for gastric neuroendocrine tumor (CEA424-SV40 T Antigen transgenic mouse model system). A significant up-regulated expression of Prap1 gene, which is the most strongly up-regulated gene identified by the array, was observed in the RAD001 resistant tumor cells both in vitro and in vivo. Preliminary experiment showed that down-regulating Prap1 expression in the RAD001 resistant gastric neuroendocrine tumor cells significantly enhanced the anti-proliferation effect induced by RAD001 treatment. With special emphasis on the molecularly targeted therapy for high grade gastric cancer with a neuroendocrine phenotype, this study aimed to clarify the possible role of Prap1 gene during the process of RAD001 resistance of neuroendocrine tumor cells, to evaluate the prospect of Prap1 as a promising target for anti-resistance strategy, and to proclaim the underlying gene regulating mechanisms involved by this gene to RAD001 resistance induced by long term treatment. This project will provide valuable preclinical experience for the treatment of neuroendocrine tumors and contribute to the establishment of more effective clinical management.

神经内分泌表型在多种肿瘤中预示着更高的恶性程度和更差的疾病预后。虽然以RAD001为代表的mTOR抑制剂能延长患者的无进展生存期，但肿瘤细胞的耐药现象却极大地削弱了药物在体内的抑癌作用。目前神经内分泌肿瘤对RAD001耐药的分子机制尚不明确。申请人前期基于一个新型神经内分泌胃癌小鼠模型，对RAD001耐药相关基因进行了筛选，发现Prap1在体内外耐药模型中的表达水平明显上调，是递增幅度最为显著的基因。进一步的预实验也发现在耐药细胞株中敲减Prap1的表达可显著增强RAD001抑制肿瘤细胞增殖的作用。本课题立足神经内分泌胃癌的靶向干预，旨在明确Prap1表达上调与RAD001耐药之间的关系，阐明在此过程中Prap1的相关功能，评估Prap1作为一个新的治疗靶点的潜在价值，并在此基础上初步探索Prap1参与诱导耐药的基因分子调控机制，为建立更科学高效的临床抑癌手段提供新的思路和临床前实验基础。

神经内分泌表型在多种肿瘤中预示着更高的恶性程度和更差的疾病预后。虽然以RAD001为代表的mTOR抑制剂能延长患者的无进展生存期，但肿瘤细胞的耐药现象却极大地削弱了药物在体内的抑癌作用。目前神经内分泌肿瘤对RAD001耐药的分子机制尚不明确。本课题基于一个新型神经内分泌胃癌小鼠模型，建立了对RAD001耐药的体内外神经内分泌胃癌模型体系。经基因芯片筛选，发现Prap1在体内耐药模型中的表达水平明显上调，是递增幅度最为显著的基因。研究在RNA及蛋白层面证实了长期RAD001治疗会导致神经内分泌胃癌细胞中Prap1水平升高，而过表达Prap1则会降低神经内分泌胃癌细胞对RAD001治疗的敏感性，促进克隆形成，抑制细胞凋亡，从而明确了Prap1表达上调与神经内分泌胃癌细胞对RAD001产生耐药之间的关系。另一方面，在已经建立的耐药细胞株RAD-424GC，RAD-mGC3，RAD-mGC8中敲减Prap1表达能增强RAD001治疗在抑制肿瘤细胞增殖和克隆形成中的作用，而在低分化胃癌人群中的Kaplan-Meier Plotter分析证实了Prap1高表达患者的中位生存期较低表达患者显著缩短，以此评估了Prap1作为一个新的治疗靶点的潜在价值。进一步的实验揭示了经RAD001长期治疗后神经内分泌胃癌组织基因表达谱的变化，阐述了细胞衰老相关信号的改变，初步阐明了Prap1参与诱导神经内分泌胃癌细胞对RAD001产生耐药的基因分子调控机制，为建立更科学高效的临床抑癌手段提供了新的思路和临床前实验基础。